Design and elucidation of an insecticide from natural compounds targeting mitochondrial proteins of Aedes aegypti.

Developing new pesticides poses a significant challenge in designing next-generation natural insecticides that selectively target specific pharmacological sites while ensuring environmental friendliness. In this study, we aimed to address this challenge by formulating novel natural pesticides derived from secondary plant metabolites, which exhibited potent insecticide activity. Additionally, we tested their effect on mitochondrial enzyme activity and the proteomic profile of Ae. aegypti, a mosquito species responsible for transmitting diseases. Initially, 110 key compounds from essential oils were selected that have been reported with insecticidal properties; then, to ensure safety for mammals were performed in silico analyses for toxicity properties, identifying non-toxic candidates for further investigation. Subsequently, in vivo tests were conducted using these non-toxic compounds, focusing on the mosquito's larval stage. Based on the lethal concentration (LC), the most promising compounds as insecticidal were identified as S-limonene (LC50 = 6.4 ppm, LC95 = 17.2 ppm), R-limonene (LC50 = 9.86 ppm, LC95 = 27.7 ppm), citronellal (LC50 = 40.5 ppm, LC95 = 68.6 ppm), R-carvone (LC50 = 61.4 ppm, LC95 = 121 ppm), and S-carvone (LC50 = 62.5 ppm, LC95 = 114 ppm). Furthermore, we formulated a mixture of R-limonene, S-carvone, and citronellal with equal proportions of each compound based on their LC50. This mixture specifically targeted mitochondrial proteins and demonstrated a higher effect that showed by each compound separately, enhancing the insecticidal activity of each compound. Besides, the proteomic profile revealed the alteration in proteins involved in proliferation processes and detoxification mechanisms in Ae. aegypti. In summary, our study presents a formulation strategy for developing next-generation natural insecticides using secondary plant metabolites with the potential for reducing the adverse effects on humans and the development of chemical resistance in insects. Our findings also highlight the proteomic alteration induced by the formulated insecticide, showing insight into the mechanisms of action and potential targets for further exploration in vector control strategies.

Insecticidal activity of essential oils from American native plants against Aedes aegypti (Diptera: Culicidae): an introduction to their possible mechanism of action.

Searching for new bioactive molecules to design insecticides is a complex process since pesticides should be highly selective, active against the vector, and bio-safe for humans. Aiming to find natural compounds for mosquito control, we evaluated the insecticidal activity of essential oils (EOs) from 20 American native plants against Aedes aegypti larvae using bioassay, biochemical, and in silico analyses. The highest larvicide activity was exhibited by EOs from Steiractinia aspera (LC50 = 42.4 µg/mL), Turnera diffusa (LC50 = 70.9 µg/mL), Piper aduncum (LC50 = 55.8 µg/mL), Lippia origanoides (chemotype thymol/carvacrol) (LC50 = 61.9 µg/mL), L. origanoides (chemotype carvacrol/thymol) (LC50 = 59.8 µg/mL), Hyptis dilatata (LC50 = 61.1 µg/mL), Elaphandra quinquenervis (LC50 = 61.1 µg/mL), and Calycolpus moritzianus (LC50 = 73.29 µg/mL) after 24 h. This biological activity may be related to the disruption of the electron transport chain through the mitochondrial protein complexes. We hypothesized that the observed EOs' effect is due to their major components, where computational approaches such as homology modeling and molecular docking may suggest the possible binding pose of secondary metabolites that inhibit the mitochondrial enzymes and acetylcholinesterase activity (AChE). Our results provided insights into the possible mechanism of action of EOs and their major compounds for new insecticide designs targeting the mitochondria and AChE activity in A. aegypti for effective and safe insecticide.

A new porphyrin as selective substrate-based inhibitor of breast cancer resistance protein (BCRP/ABCG2).

The ABCG2 transporter plays a pivotal role in multidrug resistance, however, no clinical trial using specific ABCG2 inhibitors have been successful. Although ABC transporters actively extrude a wide variety of substrates, photodynamic therapeutic agents with porphyrinic scaffolds are exclusively transported by ABCG2. In this work, we describe for the first time a porphyrin derivative (4B) inhibitor of ABCG2 and capable to overcome multidrug resistance in vitro. The inhibition was time-dependent and 4B was not itself transported by ABCG2. Independently of the substrate, the porphyrin 4B showed an IC50 value of 1.6 µM and a mixed type of inhibition. This compound inhibited the ATPase activity and increased the binding of the conformational-sensitive antibody 5D3. A thermostability assay confirmed allosteric protein changes triggered by the porphyrin. Long-timescale molecular dynamics simulations revealed a different behavior between the ABCG2 porphyrinic substrate pheophorbide a and the porphyrin 4B. Pheophorbide a was able to bind in three different protein sites but 4B showed one binding conformation with a strong ionic interaction with GLU446. The inhibition was selective toward ABCG2, since no inhibition was observed for P-glycoprotein and MRP1. Finally, this compound successfully chemosensitized cells that overexpress ABCG2. These findings reinforce that substrates may be a privileged source of chemical scaffolds for identification of new inhibitors of multidrug resistance-linked ABC transporters.

Tetrahydroquinoline/4,5-Dihydroisoxazole Molecular Hybrids as Inhibitors of Breast Cancer Resistance Protein (BCRP/ABCG2).

Multidrug resistance (MDR) is one of the major factors in the failure of many chemotherapy approaches. In cancer cells, MDR is mainly associated with the expression of ABC transporters such as P-glycoprotein, MRP1 and ABCG2. Despite major efforts to develop new selective and potent inhibitors of ABC drug transporters, no ABCG2-specific inhibitors for clinical use are yet available. Here, we report the evaluation of sixteen tetrahydroquinoline/4,5-dihydroisoxazole derivatives as a new class of ABCG2 inhibitors. The affinity of the five best inhibitors was further investigated by the vanadate-sensitive ATPase assay. Molecular modelling data, proposing a potential binding mode, suggest that they can inhibit the ABCG2 activity by binding on site S1, previously reported as inhibitors binding region, as well targeting site S2, a selective region for substrates, and by specifically interacting with residues Asn436, Gln398, and Leu555. Altogether, this study provided new insights into THQ/4,5-dihydroisoxazole molecular hybrids, generating great potential for the development of novel most potent ABCG2 inhibitors.